C4-like Sesuvium sesuvioides (Aizoaceae) exhibits CAM in cotyledons and putative C4-like + CAM metabolism in adult leaves as revealed by transcriptome analysis

Background The co-occurrence of C4 and CAM photosynthesis in a single species seems to be unusual and rare. This is likely due to the difficulty in effectively co-regulating both pathways. Here, we conducted a comparative transcriptomic analysis of leaves and cotyledons of the C4-like species Sesuvium sesuvioides (Aizoaceae) using RNA-seq. Results When compared to cotyledons, phosphoenolpyruvate carboxylase 4 (PEPC4) and some key C4 genes were found to be up-regulated in leaves. During the day, the expression of NADP-dependent malic enzyme (NADP-ME) was significantly higher in cotyledons than in leaves. The titratable acidity confirmed higher acidity in the morning than in the previous evening indicating the induction of weak CAM in cotyledons by environmental conditions. Comparison of the leaves of S. sesuvioides (C4-like) and S. portulacastrum (C3) revealed that PEPC1 was significantly higher in S. sesuvioides, while PEPC3 and PEPC4 were up-regulated in S. portulacastrum. Finally, potential key regulatory elements involved in the C4-like and CAM pathways were identified. Conclusions These findings provide a new species in which C4-like and CAM co-occur and raise the question if this phenomenon is indeed so rare or just hard to detect and probably more common in succulent C4 lineages. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10553-2.


Introduction
In a number of eudicot families, C 4 photosynthesis evolved in ancestrally succulent lineages (see [1], for an overview), prominent examples are Chenopodiaceae [2], Aizoaceae-Sesuvioideae [3], Portulacaceae [4] and Zygophyllaceae [5].Some families like Aizoaceae and Portulacaceae also use crassulacean acid metabolism (CAM) as carbon concentrating mechanism (CCM).Both CCMs share the same core metabolic enzymes, both evolved repeatedly multiple times, however, it seems that they rarely co-occur.So far, the co-occurrence of C 4 and CAM was verified for only four genera, Portulaca (Portulacaceae; [6]), Spinifex (Poaceae; [7]), Ottelia (Hydrocharitaceae; [8]) and Trianthema (Aizoaceae; [9]).Recent evidence demonstrated that C 4 and CAM are operating in the same cells in Portulaca oleracea under drought conditions [10].This integration is plausible due to several copies of core C 4 genes [i.e.phosphoenolpyruvate carboxylase (PEPC)] that are recruited for C 4 and CAM, respectively, sharing a set of biochemical reactions.Detecting the co-occurrence of C 4 and CAM is laborious, requires living collections and an experimental approach, which is why this phenomenon has not been documented very often.However, we hypothesize that it might be more common in succulent C 4 lineages than currently known.
C 4 photosynthesis is an adaptive evolutionary response to the harmful effect of photorespiration under hot and dry growing conditions, by concentrating CO 2 around RUBISCO [11].This allows for a remarkably efficient photosynthesis, as well as water and nitrogen use.The C 4 pathway is a complex combination of anatomical and biochemical specialization.In succulent C 4 lineages the C 4 anatomy is particularly diverse [1,12].Often the Kranz cells are not arranged as an inner wreath around the vascular bundles like in plants with typical Kranz anatomy, but form a continuous inner chlorenchyma layer around the central water storage tissue of the leaf (e.g., [3,13]).The general pathway in which CO 2 is converted to bicarbonate (HCO 3

−
) by carbonic anhydrase in mesophyll cells (MCs) and then fixed to the 3-carbon molecule phosphoenolpyruvate by the enzyme PEPC to form the 4-carbon molecule oxaloacetate (OAA) unifies all plants with C 4 photosynthesis.OAA is then either reduced to malate or transaminated to aspartate.After diffusing to an adjacent Kranz cell, malate or aspartate is primarily decarboxylated by the enzymes either NADP-dependent malic enzyme (NADP-ME) or NAD-dependent malic enzyme (NAD-ME).This decarboxylation releases CO 2 in high concentrations around RUBISCO and ensures high photosynthetic efficiency.This carbon concentration mechanism (CCM) is supported and facilitated by a decrease in the ratio of mesophyll to Kranz cells as opposed to the C 3 ancestors.
Unlike C 4 photosynthesis, the CCM of CAM photosynthesis is temporally asynchronous in a single-cell system.During the night, plants open stomata, and CO 2 is fixed and converted to malate, which is stored in the vacuole as malic acid.During the day, stomata are closed and stored malate is transported out the vacuole and decarboxylated to release CO 2 that is then fixed by RUBISCO and enters the Calvin cycle for sugar production.This asynchronous carbon fixation system allows plants to keep their stomata closed to avoid water loss through evapotranspiration during the hottest period of the day.Thus, plants with this type of metabolism are able to grow in hot and dry environments.While it is straight forward to detect obligate CAM plants by means of a strong carbon isotope signal and consistent differences between morning and evening acid concentrations, it is laborious to detect facultative or weak CAM plants that only induce CAM under stress [14,15].Weak CAM can neither be detected by carbon isotope ratios in C 3 species nor in C 4 species.In C 3 species, the discrimination of RUBISCO towards the heavier C isotope and in C 4 species the much higher activity of the PEPC in the C 4 pathway hides the low CAM signal.
Aizoaceae comprise annual or perennial herbs, rarely shrubs or trees, growing in tropical and subtropical regions, predominantly in South Africa [16].Most species of the family are succulent and many, especially from subfamily Mesembryanthemoideae and Ruschioideae are documented CAM plants [14].In Aizoaceae, C 4 photosynthesis is restricted to subfamily Sesuvioideae and likely evolved multiple times [3].A striking diversity of leaf anatomical types and the occurrence of both biochemical subtypes of C 4 (NAD-ME and NADP-ME) can be observed.In addition to this photosynthetic diversity, two species from Sesuvioideae have been reported to activate low CAM under drought, i.e., the C 3 species Sesuvium portulacastrum and the C 4 species Trianthema portulacastrum ( [9,15]).Yet another species of this subfamily arouses curiosity: Sesuvium sesuvioides, a succulent C 4 -like species with uncommon C 4 features and photosynthetic plasticity during leaf aging.Structural, physiological, and biochemical analysis of Sesuvium sesuvioides indicated a relatively high MC/bundle sheath cell (BSC) ratio and the presence of RUBISCO large subunit together with PEPC in the MCs [3].Furthermore, a decrease of C 4 enzyme activities was observed from young to mature to senescent leaves [17].Although Bohley et al. [17] did not observe any CAM activity under well-watered conditions, they did not exclude the existence of CAM under dry conditions.
Such species that exhibit photosynthetic variability may contain footprints left from the evolution of CAM and C 4 photosynthesis and thus provide useful information to either disentangle or gain new insight into the evolution and regulation of C 4 and CAM metabolism.From this perspective, the photosynthetic flexibility in subfamily Sesuvioideae represents an excellent potential study group.We need such models as with climate change, many agricultural regions approaching their potential peak of productivity [18], and with an estimated population of 10 billion people by 2050, C 4 and CAM represent a promising way to increase productivity and hence yield to meet global demands for food owing to their intrinsic ability to thrive in hot and dry environments.Thus, both CCMs are targets for genetic engineering into C 3 species.Substantial efforts taken in the past to introduce C 4 and CAM features into C 3 plants failed to reach the envisioned goals due to lack of knowledge of C 4 and CAM photosynthesis at the system level [19].Therefore, mechanisms underlying C 4 and CAM anatomical structure, gene-specific expression, and regulation network in C 4 must be clarified further [19], and each new mosaic stone will help to solve the conundrum.
To test our hypothesis that S. sesuvioides operates combined C 4 and CAM photosynthesis, we (1) performed a comparative transcriptome analysis between cotyledons and young leaves of S. sesuvioides (C 4 -like species) and young leaves of Sesuvium portulacastrum (C 3 species) under stressful conditions: high light intensity and drought.We then (2) investigated the integration of C 4 -like + CAM in S. sesuvioides via the identification of candidate genes linked to C 4 and CAM previously identified in Portulaca [20].Finally, we explored the regulatory elements controlling C 4 -like and C 3 pathways.Our analyses revealed that S. sesuvioides is operating weak CAM in cotyledons and C 4 -like + CAM in leaves as proved by gene expression analysis and supported by acid titration.Moreover, C 4 -like + CAM candidate genes were found up-regulated during the day suggesting the integration of C 4 -like + CAM metabolism in S. sesuvioides.

Plant materials
Plants of S. sesuvioides (C 4 -like) and S. portulacastrum (C 3 ) were grown from seeds and cuttings respectively, in the experimental greenhouse of the Munich-Nymphenburg Botanical Garden, Germany.Sesuvium sesuvioides seeds were collected from a location situated ~ 80 km east of Sendelingsdrif, Karas, Namibia [~ 28.20946°S, 17.28936°E, 208 m altitude, voucher: Klak 2431 (BOL)] and S. portulacastrum plant materials were collected in Texas, USA (MSB Serial number 0394523; year collected: 2007).For simplicity, we will sometimes use C 4 instead of C 4 -like.Germinated seedlings (about 1 cm) and oneyear-old plants of S. sesuvioides and one-year-old plants of S. portulacastrum were transferred to climate chambers with the following parameters: photoperiod light/ dark 14 h/10 h, 60% humidity, [CO 2 ] = 400 ppm, maximum light intensity = 785 μmol/m 2 /s, day/night temperature of 25/22 °C.Plants were watered every 2 days.This created stressful conditions specifically drought with changes in leaves colors as shown in the pictures (Additional file 1).Samples for transcriptome were harvested two weeks after transferring the plants to the climate chamber.Three young adult leaves of three plants of each species were collected during the day (1 pm) and for S. sesuvioides cotyledons three replicates of four plants were harvested during day (at 1 pm) and night (9 pm, one hour after the light was off ).The plant S. sesuvioides was first identified by Cornelia Klak and later confirmed by Katharina Bohley.Sesuvium portulacastrum was also identified by Katharina Bohley.Vouchers of S. sesuvioides (LS 257) and S. portulacastrum (LS 221) have been deposited at the Herbarium MSB with serial numbers M-0356568 and M-0356569 respectively.

Titratable acidity
Possibility of CAM activity under stressful conditions was investigated in cotyledons and leaves of S. sesuvioides and leaves of S. portulacastrum via comparative titratable acidity between 30 min before the end and 30 min before the beginning of the light period (19.30h and 5.30 h, respectively).Cotyledons and leaves were harvested and snap-frozen in liquid nitrogen and stored at -20 °C.Since cotyledons were small, eight plants of S. sesuvioides for each harvesting time were collected.Stored cotyledons and leaves were chopped and weighed.About 50 mg of the cotyledons and leaves material were incubated at 60 °C in 20% ethanol for 60 min.The extract obtained was aliquoted into three replicates of the same volume (1 mL).The extracted acid was neutralized by adding 0.01 M NaOH in 1ul increments [21].

RNA extraction and sequencing
Total RNA was extracted from leaves and cotyledons as described by Siadjeu et al. [22] using innuPREP Plant RNA Kit (Analytik Jena AG, Jena, Germany).Total RNA quality control was performed using the 2100 Bioanalyzer (Agilent Technologies) and Agarose gel electrophoresis.Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads.After quality control and fragmentation, the first-strand cDNA was synthesized using random hexamer primers followed by the second-strand cDNA synthesis.The library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification.

Transcriptome analysis
An overview of the bioinformatics pipeline presenting software and respective versions used is presented in Fig. 1.Sequence read quality control was assessed using FastQC (https:// www.bioin forma tics.babra ham.ac.uk/ proje cts/ fastqc/) and summarized with MultiQC [23].Random sequencing errors in reads were corrected with a k-mer-based method implemented in Rcorrector [24] and uncorrectable reads were removed from the reads using TranscriptomeAssemblyTools (Fig. 1) (https:// github.com/ harva rdinf ormat ics/ Trans cript omeAs sembl yTools).Low-quality reads and adapters were filtered using TrimGalore v0.6.7 (https:// github.com/ Felix Krueg er/ TrimG alore/ relea ses).The rRNA reads were removed by aligning trimmed reads against the SILVA v-138 rRNA database using Bowtie2 v.2.4.5 [25].De novo transcriptome assembly was performed using Trinity v.2.14.0 [26] with the following parameters (Trinity -seqType fq -SS_ lib_type RF -max_memory 200G -min_contig_length 300 -CPU 16).The de novo transcriptome assembly quality was first confirmed by aligning cleaned reads back to the corresponding de novo transcriptome assembly using Bowtie2 v.2.4.5.Secondly, the BUSCO score (odb10), which evaluates the completeness of the transcriptome, was determined using BUSCO v4 [27].For the downstream analysis, the initial assembly of each species was processed as follows.We reduced the transcriptome data by clustering transcripts with 98% similarity with CD-HIT v4.8.1 [28].Then, we selected only transcripts harboring coding sequences with TransDecoder v.5.5.0 (https:// github.com/ sghig none/ Trans Decod er).Trans-Decoder performs a precomputed blastX alignment to the Uniprot protein sequence database to improve the prediction of open reading frames.Finally, we used the indexes of transcripts containing coding sequences to subset the initial assemblies.The reduced transcriptomes were used for differential expression analysis.For differential expression analysis, we exclusively focused on S. sesuvioides when comparing leaves collected during the day to cotyledons collected both during the day and at night.For the comparison between C 3 and C 4 -like species, we used young adult leaves collected during the day.

Transcript quantification and differential expression analysis
We quantified transcript abundance with Salmon by aligning the reads of each species to its reduced transcriptome (Fig. 1).The software tximport v. 1.24.0 [29] was used to import transcript level abundances, estimated counts, and effective lengths for differential expression analysis.For comparison between the leaves of C 3 and C 4 -like species, we searched for orthologs between their reduced transcriptomes using OrthoFinder v2.5.4 [30].Based on the orthology between C 3 and C 4 -like species, we obtained unique indexes by blasting C 3 and C 4 -like species transcripts against each other.The best blast hits with the lowest e-value and high bit scores were selected and considered homologous.Transcripts of the C 4 -like species were used as a reference for the unique ID.If a C 4 -like species transcript was hit multiple times with transcripts of the C 3 species, only one randomly selected transcript was kept to get a similar number of transcripts for differential expression analysis.Finally, the unique C 4 -like ID was changed in the Salmon output of the C 3 species before importing the data with tximport (Script is available at https:// github.com/ Siadj eu/ Sesuv ioide ae_ C4-CAM).We assessed differential expression with the program Deseq2 [31].Transcripts with p-value and p-adjusted as false discovery rate < 0.05 were considered significantly expressed andLog2FC was set > 1.

Pathway and gene ontology (GO) annotation
Metabolic pathways and annotations of differentially expressed (DE) transcripts were assigned via the tool Mercator4 [32].Swiss-prot protein sequences database and prot-scriber were included to improve the annotations.Unassigned transcripts were manually assigned based on the knowledge of the molecular functions in C 4 /CAM, photorespiration, and starch metabolism.We assigned GO terms to DETs using Blast2GO through OmicsBox with cutoff = 55, GO weight = 5, e-value = 1.e-5,HSP-hit coverage cutoff = 80 and hit filter = 500.We enriched the GO terms using Fisher's exact test via OmicsBox.

Phylogenetic analysis of PEPC isoforms
To investigate whether various isoforms of PEPCs are involved in CAM or C 4 photosynthesis, we conducted a phylogenetic analysis of PEPC derived from our study alongside three distinct genes (PPC-1E1, PPC-1E2 and PPC-2) that encode PEPC from 35 different species.It has been found that PPC-1E1 is consistently involved in both CAM and C 4 photosynthesis [20].We obtained the protein sequences of these genes from the UniProt database (https:// www.unipr ot.org/, accessed on 06.03.2024).To generate proteins from the transcript sequence of PEPC discovered in our study, we employed TransDecoder v5.7.1 (https:// github.com/ Trans Decod er/ Trans Decod er), and extracted the longest open reading frames of these transcripts.We constructed a multiple sequence alignment using MAFFT v.7.520 [33], and subsequently built an unrooted maximum likelihood tree using RAxML v8.2.13 [34].

Co-expression network analysis
The co-expression analysis was performed using the unsupervised machine learning algorithm k-means in R. We used the three most popular methods for determining the optimal cluster: the Elbow and silhouette [35] methods and gap statistic [36].The normalized read counts of DETs were used.The maximum number of clusters (k) was set to 10.If transcription factors (TFs) and phytohormones were clustered with C 4 -like or CAM genes, they were considered candidate TFs and phytohormones controlling C 4 -like or CAM photosynthesis.The k-means clustering script is available underhttps:// github.com/ Siadj eu/ Sesuv ioide ae_ C4-CAM.

Titratable acidity
To investigate CAM induction in leaves and cotyledons of S. sesuvioides and leaves of S. portulacastrum, we conducted titratable acidity tests.Interestingly, the tests revealed significant overnight acid accumulation in leaves (t-test, adjusted p-value = 0.001) and cotyledons (t-test, adjusted p-value = 0.018) of S. sesuvioides and in leaves of S. portulacastrum (t-test, adjusted p-value = 0.018) (Fig. 2).Variations in nocturnal acidification were about 7 μmol/g fresh weight of free acids (FA) in cotyledons of S. sesuvioides, and 22 and 20 μmol/g FA in leaves of S. sesuvioides and S. portulacastrum, respectively.Similar overnight acid accumulation has been reported in the leaves of S. portulacastrum under drought stress [40].Moreover, acid accumulations of 15 and 18 ∆H + have been observed in drought stress in Portulaca amilis and P. oleracea, respectively, which induce CAM photosynthesis under stress conditions [41].These differences in acidity between the morning and night indicate a weak CAM induction.Therefore, we analyzed the transcriptome profiles of S. sesuvioides and S. portulacastrum in relation to C 4 and CAM photosynthesis.

Differential expression analysis across Sesuvium species
Two comparative analyses were carried out: (1) between leaves and cotyledons of S. sesuvioides, and (2) between leaves of S. sesuvioides and leaves of S. portulacastrum.
Leaves and cotyledons of S. sesuvioides were clearly separated based on their expression profile (Fig. 3A).Likewise, C 3 and C 4 -like species were clustered according to their photosynthetic type mainly along PC1 (Fig. 3B).A total of 6,063 transcripts were found to be significantly DE between leaves and cotyledons of S. sesuvioides during the day (L and CD), of which 2,492 were up-regulated in leaves and 3,571 in cotyledons (Fig. 3C).When comparing leaves during the day (L) and cotyledons of S. sesuvioides during the night (CN), 1242 were up-regulated in leaves and 2,941 in cotyledons.Comparison of cotyledons between night (CN) and day (CD) revealed that 1,706 and 821 transcripts were found to be up-regulated at night and day, respectively.Between the C 3 (S.portulacastrum) and C 4 (S.sesuvioides) species, we found 20,867 orthologous transcripts in a 1:1 relationship.Out of these orthologs, 3,860 transcripts were significantly up-regulated in the C 3 species and 2,433 in the C 4 -like species (Fig. 3C).

Functional annotation of DETs between S. sesuvioides and S. portulacastrum
Functional annotations of DETs to land plant protein sequences were assigned using Mercator4.To explore the difference between ancestral C 3 photosynthesis to C 4 -like photosynthesis in S. sesuvioides, we compared the expression profile of S. portulacastrum (C 3 ) and S. sesuvioides (C 4 -like) with respect to CCM.According to photosynthetic sub-pathways, DETs were clustered and the number of DETs associated with each pathway was plotted (Fig. 4).We found a significant accumulation of genes involved in C 4 -related pathways (except PEP regeneration that was only found in the C 4 -like species) in both species (Fig. 4A, Additional file 2).The number of genes involved in carboxylation, proton pump, transfer acid generation, and transporter were higher in S. sesuvioides (C 4 -like; Fig. 4A, Additional file 2).Conversely, genes related to decarboxylation and photorespiration were abundant in the C 3 species as compared to C 4 -like species.
We then investigated genes related to carboxylation and decarboxylation.Surprisingly, while PEPC1 (TRIN-ITY_DN0_c3_g1_i12) was up-regulated in the C 4 -like species, PEPC3 (TRINITY_DN9611_c0_g1_i3) and PEPC4 (TRINITY_DN14827_c0_g1_i3) were significantly expressed in the C 3 species (Fig. 4B).PPDK and PPCK1 were up-regulated in the C 4 -like species (Additional file 3).The decarboxylation enzymes chloroplastic NADP-ME4, NADP-ME, and NAD-ME were significantly accumulated in the C 3 species.However, another NADP-ME copy was up-regulated in the C 4 -like species.These findings suggest that S. sesuvioides as C 4 -like species employs NADP-ME as a decarboxylation enzyme but can additionally use NAD-ME (Additional file 3).

Functional annotation of DETs between leaves and cotyledons of S. sesuvioides
We examined the process of photosynthesis in the leaves and cotyledons of S. sesuvioides.We identified DE transcripts (p < 0.05, log 2 FC > 1) related to CCM according to annotation (Fig. 6, Additional file 2).The transcripts related to CCM were subsequently clustered based on their role in decarboxylation, Calvin cycle, carboxylation, citrate generation, PEP regeneration and regulation, photorespiration, transfer acid generation, and transporter.We then counted the number of related transcripts of each cluster to identify the photosynthetic mode of cotyledons and leaves (Fig. 6A, Additional file 2).We found that transcripts involved in Calvin cycle, photorespiration, carboxylation, and decarboxylation were abundant in cotyledons as compared to leaves, whereas transporters involved in CCMs (C 4 and CAM), transfer acid generation and PEP regeneration were higher in leaves than in cotyledons (especially those collected during the day).This suggests that different CCMs are acting in leaves and cotyledons of S. sesuvioides.To investigate further, we plotted the abundance of transcripts associated with the functional categories.CAM differs from C 4 by a nocturnal CO 2 fixation and accumulation of malate or citrate in the vacuole, and a diurnal decarboxylation of accumulated malate by the malic enzymes (e.g.NADP-ME).Our results showed that transcripts encoding carboxylation enzymes (PEPC4 and PPCK1) were upregulated in leaves as compared to cotyledon collected during the day (CD), while transcripts encoding decarboxylation enzymes NADP-ME and chloroplastic NADP-ME4 were significantly abundant in cotyledons collected during the night (CN) (Fig. 6B).Moreover, phylogenetic analysis showed that PEPC4 (TRINITY_DN14827_c0_g1_i3) grouped with a PPC-1E1 gene, along with several PPC-1E2 and PPC-2 genes (Additional file 5).These results indicate a higher decarboxylation rate in CD and CN.Thus, we suspected the possibility of CAM and C 4 -like photosynthesis in cotyledons and leaves of Sesuvium sesuvioides, respectively.
To determine whether CAM is occurring in the cotyledons, we compared the transcript profiles of CD and CN (Fig. 6A).In CAM plants, malate generated at night is supposedly transported to the vacuole by a malate transporter aluminium-activated malate transporter (ALMT) [42].We observed a significant increase in transcripts of ALMT2 in CN (Fig. 6B).The vacuolar malate influx is driven by the difference in membrane potential established by vacuolar-type proton adenosine triphosphatase ATPase (VHA) and the pyrophosphate-energized membrane proton pump (AVP) [43].However, as in Portulaca [10], no significant expression of these genes was observed in the CN.Although no significant expression of decarboxylation enzymes NADP-ME in CN when compared to CD, a mitochondrial NAD-ME was highly abundant in CN (Fig. 6B).However, we found that RUBISCO small unit (RBCS1) (Additional file 3) was upregulated in CD.This suggested that the decarboxylation activity of NAD-ME at night is aimed primarily at malate respiration [44].Additionally, ATP-citrate synthase alpha chain protein 3 (ACL-3) which is involved in citrate synthesis [45] was found up-regulated in CN.Cotyledons showed lower citrate levels at 11 am compared to leaves of S. sesuvioides (Siadjeu, unpublished data).Citrate is produced when acetyl CoA reacts with OAA suggesting that citrate contributes as well to the acidification in S. sesuvioides at night.Facultative CAM species such as Talinum triangulare exhibit increased citrate levels during the night [46].
Another feature of CAM photosynthesis is the nightly regeneration of PEP via phosphorolytic starch degradation [47,48].Moreover, Moreno-Villena et al. [10] hypothesized that CAM induction is associated with the increase in sugar transporter.The number of transcripts related to starch/sugar transporter and starch phosphorolytic degradation was high in CN (Fig. 6C, Additional file 2).We found that genes encoding the probable alphaamylase 2 (AMY2), alpha-amylase (AMY) and alpha-1,4 glucan phosphorylase L-2 isozyme (PHO2) involved in starch phosphorolytic degradation were significantly abundant at night (Fig. 6D, Additional file 2).The sugar transporters, SWEET14, SWEET17, ERD6-like 5 were up-regulated at night (Fig. 6D).It is worth mentioning that we did not observe a significant expression of PEPC at night.However, pyruvate phosphate dikinase (PPDK) was significantly abundant in the cotyledons at night (Fig. 3D).The enzyme PPDK catalyzes the regeneration of the CO 2 acceptor PEP via pyruvate [49].

Regulation and hormonal signaling in Sesuvium
To identify potential regulation and signaling elements, we performed an unsupervised k-means clustering.The three methods used showed the best k was two (Additional file 6).We found that almost all transcripts involved in photosynthesis, starch metabolism, transcription factor, and phytohormone signaling were grouped together (Cluster with the highest number of transcripts) for all comparisons (Additional files 7-8).To identify possible candidates involved in the regulation of the CCMs studies, we clustered TFs found in groups including photosynthesis, starch metabolism, and phytohormone signaling according to their families (Fig. 7, Additional file 2).
For comparison between S. sesuvioides and S. portulacastrum, TF families C2C2, C2H2, GRAS, HOMEOBOX, MYB-related, NAC, TEOSINTE BRANCHED1/ CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP), WRKY, basic HELIX-LOOP-HELIX (bHLH), and basic LEUCINE ZIPPER (bZIP) were found on top of the list (Fig. 7A, Additional file 2).Except for TFs from the GRAS family present only in C 4 -like, all others were found in C 4 -like and C 3 species (Additional file 3).We selected based on the literature candidate TFs that are potentially involved in the regulation of C 4 and CAM (Fig. 7B).Different isoforms are recruited in the regulation of C 3 and C 4 -like species.Our data showed that REVEILLE 8, bHLH143, SCL6, COL5, and DOF1.2 were significantly up-regulated in C 4 -like species, whereas REVEILLE 6, COL15, NFYA7, and HB6 were up-regulated in the C 3 species (Fig. 7B, Additional file 3).We identified 63 nonredundant motifs (p < 0.01, E-value < 0.05) in sequences of genes upregulated in the C 4 -like species while 36 motifs were found in the C 3 species (Additional file 9).The top five of the most enriched motifs were annotated to C2C2-DOF families in C 4 -like and the C 3 species with the element CTT TTT (Table 2).Although the most enriched motifs were similar between the C 4 -like species and the C 3 species, the second most enriched motifs were elements (GAGA , BBR/BPC family) and (CAC CAA CM, MYB family) in the C 4 -like and C 3 species, respectively.We found several motifs often present in the same transcript sequences.This suggests a coordinated and regulatory network of TFs controlling photosynthesis in which motif CTT TTT is dominant.
Our results indicated that the same TF families are recruited to a variable degree for controlling cotyledons and leaves of S. sesuvioides.The top 10 TF families abundant in cotyledons and leaves during the day and in CN were bHLH), C2C2, APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF), WRKY, MADS/ AGL, bZIP, NAC, MYOLOBLASTOSIS (MYB)-related, GARP (GOLDEN2, ARR-B, PSR1), and HOMEOBOX (Fig. 7C, Additional file 2).While during the day, the number of TFs related to C2C2, bHLH, and bZIP families were higher in leaves than in cotyledons, no TFs related to HOMEOBOX and AP2/ERF were enriched in leaves.When comparing CD and CN, we found nearly all TF families were abundant at night.We then specifically looked at TFs that are frequently expressed in these families.Genes were selected by their potential involvement in the regulation of CAM and C 4 -like photosynthesis.Our data showed the TFs bHLH87, REVEILLE1, WRKY40, NAC83, and SCL15 were up-regulated in the CN, whereas TFs BBX24, and SCL14 were significantly abundant in the leaves (Fig. 7D).To confirm whether these TFs regulate C 4 -like and CAM photosynthesis, we explored TF binding sites in sequences of transcripts related to C 4 -like photosynthesis and starch metabolism (Table 2).Motif enrichment analysis revealed elements AAA AAG and CTT TTT from the C2C2-DOF family were the most enriched in leaves during the day, while element CTT TTT was the most enriched in cotyledons (Table 2).When comparing CD and CN, element CGC CGC C from the AP2/ERF family was enriched in CD whereas element (CTT TTT ) was enriched in all comparisons.
Phytohormones play critical roles in photosynthesis regulation and developmental processes ranging from organ initiation to senescence [50].Moreover, phytohormones have been shown to mediate TF action in C 4 and CAM plants [51].When comparing S. sesuvioides to S. portulacastrum, we found that signaling peptides, auxin, cytokinin, abscisic acid, and brassinosteroid were the top five endogenous signaling hormones (Fig. 8A, Additional file 2).There were many genes related to hormonal signals that were specific to either C 3 species or C 4 -like species (Additional file 3).For instance, genes PHP2 (cytokinin), RALF4 (signaling peptide), and IAA9 (auxin) were significantly accumulated in the C 3 species while EIR3 (auxin), ERF118 (cytokinin) and CEPR2 (signaling peptide) were significantly accumulated in the C 4 -like species (Fig. 8B).When comparing leaves to cotyledons, signaling peptides, auxin, cytokinin, jasmonic, and abscisic acids were the most abundant signaling hormones (Fig. 8C, Additional file 2).We found that the expression of CYP707A4 (abscisic acid), LHW (cytokinin), and JAR6 (jasmonic acid) increased significantly in abundance in cotyledons at night (Fig. 8D).In CD, MS17 (Auxin) and GAST1 (signaling peptide) were up-regulated, while IAA14 (auxin) and GASA1 (signaling peptide) were significantly accumulated in the leaves.Many other signaling protein genes were found specific to leaves and cotyledons and are listed in Additional file 3.
Intriguingly, when comparing leaves of the adult plants with cotyledons of S. sesuvioides, the main carboxylation enzyme was PEPC isoform 4 (PEPC4) in leaves.There are several plant PEPC copies that are classified into photosynthetic (C 4 and CAM) and nonphotosynthetic isoforms [52].In eudicots, there are three distinct lineages that encode PEPC, which are called PPC-1E1, PPC-1E2, and PPC-2 [20].According to the Uniprot database, these genes are involved in CO 2 fixation and the tricarboxylic acid cycle.Phylogenetic analysis of PEPC4 with these three genes from 35 species (Additional file 5) showed that PEPC4 (TRINITY_DN14827_c0_g1_i3) clustered together with a PPC-1E1, as well as several PPC-1E2 and PPC-2 genes.However, the presence of PPC-1E1 in this group suggests that PPC4 may be involved in C 4 photosynthesis.Indeed, PEPC4 isoform identified and up-regulated in the leaves is homologous to Arabidopsis AtPEPC4 which is involved in photosynthesis (https:// www.unipr ot.org/ unipr otkb/ Q8GVE8/ entry) and may indicate a similar role in S. sesuvioides.In line with this result, PEPC3 and PEPC4 were significantly expressed in adult C 3 leaves of S. portulacastrum when compared to the leaves of C 4 -like S. sesuvioides.PEPC3 (TRINITY_ DN9611_c0_g1_i3) was also found clustered in a similar group with PEPC4.This could also be explained by the fact that the C 3 species S. portulacastrum induces weak CAM photosynthesis under drought stress [15].Heat and drought stresses repress nitrogen metabolism enzymes [53].Thus, regulation of nitrogen metabolism is crucial to maintain plant growth under stress conditions.PEPC plays a crucial role in C 4 photosynthesis and in modulating the balance of carbon and nitrogen metabolism in Arabidopsis [54].This indicates that the up-regulation of these PEPC copies may be important to maintain the photosynthetic system and plant growth of Sesuvium species under adverse conditions.The biological role and localization of these PEPC copies in Sesuvium need to be further investigated, however, our results indicate that different PEPC copies are optimized for different photosynthetic functions in leaves and cotyledons of Sesuvium.Generally, there are three subtypes of C 4 photosynthesis depending on the decarboxylation enzymes.The main decarboxylation enzyme in S. sesuvioides was NADP-ME.The up-regulation of NADP-ME indicates that S. sesuvioides is employing NADP-ME in its decarboxylation mechanism.This result is consistent with biochemical and physiological observations in S. sesuvioides [17].Moreover, in the C 3 species, multiple copies of the chloroplastic decarboxylation enzymes NADP-ME and NAD-ME were significantly accumulated.These findings may indicate a possibility of CAM induction in S. portulacastrum.
It was shown indeed that S. portulacastrum is capable of inducing CAM in stressful conditions [15,55].ALMT9 and ALMT4 were significantly up-regulated in S. sesuvioides and S. portulacastrum, respectively.ALMT was originally responsible for nocturnal malate accumulation caused by an inward-rectifying anion-selective channel that forces only malate influx to the vacuole [56].However, Meyer et al. [57] demonstrated that AtALMT6 functions as a malate influx or efflux channel depending on the tonoplast potential.This indicates diurnal vacuolar malate efflux in both species, hence the possibility of weak CAM being induced in S. sesuvioides and S. portulacastrum.

Photosynthetic plasticity in Sesuvium
Plants exhibit plasticity for a wide variety of ecologically important traits to adjust to environmental changes [58].Photosynthetic plasticity underpins the ability of plants to acclimate and grow in adverse environments and may depend on plant ontogeny.Our data provide evidence of photosynthetic plasticity in S. sesuvioides with C 4 -like and CAM photosynthesis in leaves and cotyledons, respectively.During the day, decarboxylating enzymes were more strongly expressed in cotyledons compared to leaves while carboxylating enzymes were strongly expressed in leaves.This result was further confirmed by titratable acidity, which showed a significant accumulation of acids overnight in cotyledons.The co-occurrence of C 4 and CAM photosynthesis has already been reported in other Aizoaceae, namely for Trianthema portulacastrum [9], but this is the first time to report ontogenetic variability with respect to photosynthesis in the Aizoaceae family with CAM in cotyledons and C 4 -like in leaves.In Amaranthaceae (incl.Chenopodiaceae), Lauterbach et al. [59] based on RNA expression profiles showed the transition from C 3 photosynthesis in cotyledons to C 4 photosynthesis in adult leaves of Salsola soda.This phenomenon seems to occur in several species of Salsoleae according to C 3 -like features such as lower carbon isotope ratios and lack of Kranz anatomy in cotyledons (e.g., [60,61]).However, these species have never been tested for CAM metabolism.The presence of CAM in cotyledons may be induced by environmental cues.Indeed, no CAM was observed in the cotyledons and leaves of S. sesuvioides under well-watered conditions [17].In the climate chamber, stressful conditions were mainly created by the maximum light intensity.CAM induction has been linked to a photoprotective role in Portulaca oleracea [51].This suggests a photoprotective role of CAM induction in cotyledons of S. sesuvioides.It is worth mentioning that a significant expression of PEPC (Phosphoenolpyruvate carboxylase) was not observed in CN.This is likely due to the sampling time (one hour after the light was turned off ).However, a significant abundance of PPDK was observed in CN.This enzyme catalyzes the regeneration of PEP via pyruvate, which serves as a CO 2 acceptor.

Integration of C 4 -like and CAM photosynthesis
Our data suggested a possible co-occurrence of C 4 -like and CAM photosynthesis in a single leaf of S. sesuvioides under adverse conditions (Fig. 5, Fig. 6A).In CAM photosynthesis, nocturnally accumulated malate is translocated out of the vacuole by a malate channel for subsequent decarboxylation during the light period.In P. oleracea, a C 4 species that performs CAM when drought-stressed [10], AtALMT9 that has been associated with CAM function [62] is a vacuolar malate channel [63].Interestingly, we found ALMT9 was significantly abundant during the light period in S. sesuvioides.Thus, the up-regulation of ALMT9 in leaves of S. sesuvioides, suggests that ALMT9 may function as a vacuolar malate efflux channel in S. sesuvioides and is therefore linked to CAM function.
Taking all results together, this may imply the integration of the hybrid system C 4 -like + CAM in S. sesuvioides under stress conditions.However, the modularity of this integration needs to be investigated.This co-occurrence of C 4 -like and CAM in a single leaf in S. sesuvioides is probably facilitated by the particular C 4 -like phenotype of S. sesuvioides leaves where Rubisco is present in the MCs.The MCs of S. sesuvioides are succulent and outnumber the Kranz cells by two-fold.When the leaves grow older, the M portion becomes even larger and the carbon isotope ratios drop [17].This might indicate a photosynthetic plasticity towards a higher proportion of CAM and or C 3 relative to C 4 -like in older leaves depending on the growing conditions.

Regulation of photosynthesis and hormonal signaling in Sesuvium
Plants have the ability to choose different photosynthetic pathways, which is controlled by TFs.Six TF families i.e., C2C2, HOMEOBOX, NAC, WRKY, bHLH, and bZIP were found up-regulated in leaves and cotyledons of S. sesuvioides when compared leaves to cotyledons and also when compared to adult leaves of S. sesuvioides and to adult leaves of S. portulacastrum.These families have been hypothesized to be involved in the regulation of C 4 and CAM photosynthesis in Chenopodiaceae, Aizoaceae, and Asteraceae [51,64].However, TFs from the C2C2, and bHLH families were the most expressed in leaves, and cotyledons during day and night.Likewise, these TFs were predominant in the C 3 and C 4 -like species.This may indicate the significant weight of the C2C2 and bHLH TF families in the regulation of cotyledons and leaf development in S. sesuvioides and S. portulacastrum.The binding site of TF C 4 ZINC FINGER-TYPE (DOF3.6)from the DOF/C2H2 was most enriched in C 4 and CAM genes in all comparisons.In addition, at least three copies of DOFs were among the top five.Several copies of DOF proteins (DOF1 and DOF2) were found involved in the regulation of the light-dependent C 4 gene PEPC in maize with antagonist effects.While DOF1 activates C 4 genes, DOF2 can activate or repress them [65].These results indicate that different copies of DOF genes are likely involved in the regulation of C 4 -like and CAM genes in S. sesuvioides and S. portulacastrum, as well.It is worth mentioning that transcription factors (TFs) from the MYB, NUCLEAR FACTOR Y (NF-Y) and NAC families have been suggested to play a role in regulating CAM in facultative CAM species such as Mesembryanthemum crystallinum [66] and T. triangulare [67].While the isoforms of these genes may differ, these TF families were found to be up-regulated when comparing CAM-induced (CD) and non-induced (CN) conditions, as well as when comparing day-time leaves and cotyledons.
Transcription factors are regulated by phytohormones (signaling molecules) under environmental stresses [68].Our data showed that phytohormones were clustered with C 4 and CAM genes, as well as TFs which indicates a regulatory network involving TFs and phytohormones.Indeed, Ferrari et al. [51] found that ABA and CK-related genes regulate TFs connected to CAM and C 4 photosynthesis in Portulaca oleracea.It has been suggested that ABA plays a role in responding to drought stress in facultative CAM species such as M. crystallinum [69] and T. triangulare [46,67].In S. sesuvioides cotyledons, transcripts of genes that encode for ABA were found to be more enriched in CD as compared to CN.As the temperature at night was cooler than during the day, this could indicate that ABA might also play a role in CAM induction.While ABA and CKs have been studied intensely in CAM and C 4 photosynthesis, several other phytohormones that regulate photosynthesis (reviewed by [50]) have received little attention.Here, we found that transcripts encoding signaling peptides were the most abundant plant hormones during the day and at night in cotyledons as opposed to leaves.Similarly, diurnal and nocturnal comparison expression in cotyledons showed that signaling were predominantly accumulated.Transcripts of genes encoding for the signaling peptides Gibberellic acid (GA)-STIMULATED ARABI-DOPSIS/GA-STIMULATED TRANSCRIPT (GAST) and the RAPID ALKALINIZATION FACTOR (RAFL) were predominantly expressed in cotyledons as compared to leaves.These plant hormones play important roles in plant growth, development, and stress responses ( [70,71]), and may control cotyledon growth and response to environmental conditions in S. sesuvioides.Conversely, in leaves as opposed to CD, the most dominant hormone was jasmonic acid followed by auxin and cytokinin CKs.Jasmonic acid, auxin, and cytokinin are classical phytohormones that regulate various aspects of plant growth and abiotic and biotic stress responses.While jasmonic acid can regulate stomatal closure and opening under drought stress in Arabidopsis [72], auxin coordinates cell division, expansion, and differentiation [73], and CKs are implicated in cell cycle progression [74].Several genes encoding for these phytohormones were found to be significantly accumulated (Additional file 3) and should be used as candidate genes involved in the regulation of CCMs in these species.

Conclusions
This study of gene expression profiles of S. sesuvioides provides evidence of extraordinary photosynthetic plasticity under adverse conditions with induced CAM in cotyledons and an integration of CAM and C 4 -like photosynthesis in adult leaves.However, the modularity of the co-occurrence of the two CCMs needs to be explored in future studies.We assume that further detection of co-occurring CCMs is just a matter of more experimental studies that explicitly look for this and we believe that it is more common in succulent C 4 lineages than currently known.Our findings suggest a complex regulatory network involving TFs and phytohormones and underpin the regulation of CCMs and adaptation of Sesuvium species which grow in disturbed and highly dynamic environments.

Fig. 1
Fig. 1 Workflow of transcriptome data analysis.The blue color represents the software used.L: leaves, C: cotelydons

Fig. 2
Fig. 2 Boxplots of morning-night differences in titratable acidity of cotyledons (C) and leaves (SS) of S. sesuvioides and leaves of S. portulacastrum (SP).Values in the graph indicate the adjusted p values of significant differences between morning and night.FA: refers to free acidity, which is measured based on a normalization against the fresh weight of a sample

Fig. 4 Fig. 5
Fig. 4 Functionally annotated DETS between C 3 and C 4 species.A Stacked bar charts showing the number of functional annotated DETS involved in CCM between C 3 and C 4 species.B Abundance of selected DETS involved in CCM between C 3 and C 4 species.C Stacked bar charts showing the number of functional annotated DETS involved in starch metabolism between C 3 and C 4 species.D Abundance of selected DETS involved in starch metabolism between C 3 and C 4 species.The stacked bar charts display all transcripts that were differentially expressed

Fig. 6
Fig. 6 Functional annotated DETS between leaves (L) and cotelydons (C) of S. sesuvioides.A Stacked bar charts showing the number of functional annotated DETS involved in CCM between L and C of S. sesuvioides.B Abundance of selected DETS involved in CCM between L and C of S. sesuvioides.C Stacked bar charts showing the number of functional annotated DETS involved in starch metabolism L and C of S. sesuvioides.D Abundance of selected DETS involved in starch metabolism L and C of S. sesuvioides.The stacked bar charts display all transcripts that were differentially expressed

Fig. 7
Fig. 7 Functional annotated expressed genes related to TFs.A Stacked bar charts showing the number of functional annotated DET related to TFs between C 3 and C 4 species.B Abundance of selected DET related to TFs between C 3 and C 4 species.C Stacked bar charts showing the number of functional annotated DET related to TFs between (L) and cotelydons (C) of S. sesuvioides.D Abundance of selected DET related to TFs between L and C of S. sesuvioides.BBX: B-BOX, bZIP: basic LEUCINE ZIPPER, CEPR: C-TERMINALLY ENCODED PEPTIDE RECEPTOR, COL: CONSTANS-LIKE, HB: HOMEOBOX, NAC (NAM, ATAF and CUC), NFYA: NUCLEAR TRANSCRIPTION FACTOR Y SUBUNIT ALPHA, SCL: SCARECROW-LIKE.The stacked bar charts display all transcripts that were differentially expressed

Fig. 8
Fig. 8 Functional annotated differentially expressed genes related to phytohormones.A Stacked bar charts showing the number of functional annotated DET related to phytohormones between C 3 and C 4 species.B Abundance of selected DET related to phytohormones between C 3 and C 4 species.C Stacked bar charts showing the number of functional annotated DET related to phytohormones between (L) and cotelydons (C) of S. sesuvioides.D Abundance of selected DET related to phytohormones between (L) and cotelydons (C) of S. sesuvioides.CEPR: C-TERMINALLY ENCODED PEPTIDE RECEPTOR, CYP707A4: CYTOCHROME P450, EIR: ETHYLENE INSENSITIVE ROOT, ERF: ETHYLENE-RESPONSIVE ELEMENT BINDING

Table 1
Statistics of assembly and alignment for transcriptomes of Sesuvium sesuvioides and S. portulacastrum

Table 2
Top five motif enrichments of differentially expressed genes related to CCM (Carbon Concentrating Mechanism) and starch metabolism in Sesuvium species